Project Summary Nanoparticles offer great promise for imaging cancer and improving patient outcomes, with applications ranging from early diagnosis to early monitoring of tumor response to therapy. However, there are a number of challenges in translating nanoparticle-based contrast agents to clinical cancer imaging that do not apply to small molecule agents, most notably non-target effects and resultant tissue toxicity. We have redesigned the strategy of utilizing nanoparticles for cancer imaging from the bottom-up, avoiding the limitations of nanoparticles while harnessing their great promise: we can build nanoparticles on-demand in tumor tissue with specially engineered self-assembling small molecules. These small molecules are masked by capping groups that prevent their self-assembly until acted upon by a target endogenous enzyme, so that upon specific unmasking they form nanoparticles in the immediate enzyme vicinity. By tagging the small molecules with a radionuclide like 18F, the location and amounts of these aggregates can be detected through PET imaging. In this way, masked probe will be rapidly cleared from the body, where self-assembled nanoparticles will be retained for longer periods of time to signal target enzyme activity. By targeting caspase-3, an enzyme activated by effective cancer chemotherapy that results in the death of the tumor cell and eradication of the tumor, we can effectively detect non-invasively where the tumor is dying and early after treatment. In this project, we will be utilizing this novel strategy of controlled in situ self-assembly to monitor the response of lung cancer to chemotherapy, however with two key modifications to the performance of the probe. Firstly, we will endeavor to enhance the amount of masked small molecule probe that reaches the tumor cells through the attachment of tumor homing groups. We will explore receptor-mediated endocytosis through folate targeting, active transport through glucose targeting, and translocation through a tumor-specific cell penetration peptide independent of receptors or transporters. We hypothesize that the more small molecules reach the tumor, the higher our sensitivity will be for detecting therapy-induced tumor death. Secondly, we will limit the length of time the self-assembled nanoparticles reside in dying tumor tissue by building controlled-degradation chemistry into the small molecule. By inducing the nanoparticles to disassemble into small molecule units in a controlled fashion after imaging has been performed, tissue toxicity associated with extended nanoparticle retention that could otherwise limit clinical translation will be avoided. In building on our controlled self-assembly strategy to improve sensitivity and limit any toxic potential, we endeavor to move our imaging technology to the clinic, providing a means for personalized therapy selection and early monitoring to ultimately improve human health outcomes.